Development of a time-resolved fluorometric assay for the high throughput screening of melanin concentrating hormone receptor antagonists.

INTRODUCTION: Melanin concentrating hormone is an orexigenic hypothalamic neuropeptide, which plays an important role in the complex regulation of energy balance and body weight mediated by the melanin concentrating hormone receptor subtype 1 (MCH1). Compelling pharmacological evidence implicating MCH1 signaling in the regulation of food intake and energy expenditure has generated a great deal of interest by pharmaceutical companies as MCH1 antagonists may have potential therapeutic benefit in the treatment of obesity and metabolic syndrome. METHODS: Although radioligand receptor binding assay has been one of the most powerful tools for receptor research and drug discovery, the limitations of radioisotopes and the problems related to safety and waste disposal limits their application in high throughput screening and has led to a growing interest in alternative, nonradioactive technologies. To develop a sensitive and reproducible assay system for MCH1, the time-resolved fluorescence (TRF) receptor binding assay with AcroWell filter plates was tested and validated. RESULTS: Comparing to the radioligand receptor binding assay for MCH1, the TRF assay presented higher Z/Z' factors with the lower signal-to-noise ratio. The known high-affinity MCH1 receptor antagonist, SNAP-7941, exhibited an IC50 value of 1.66+/-0.10 nM that is very similar to the IC50 value of MCH in a radioligand binding assay with an excellent correlation coefficient (0.9884). DISCUSSION: These results suggest that our TRF receptor binding assay for MCH1 can achieve the desired sensitivity and reproducibility to replace the radioligand receptor assay in a fluorometric system that can be developed for high throughput screening.

Melanotropin receptors of murine melanoma characterized in cultured cells and demonstrated in experimental tumors in situ.

Cultured melanoma cells are known targets for the pigment-inducing actions of melanotropins such as alpha-melanocyte-stimulating hormone (alpha-MSH). The objectives of the present studies were to determine the binding properties and functional relevance of MSH binding sites in a mouse melanoma cell line and to determine whether MSH receptors are expressed in situ. The binding properties of MSH receptors in intact cells of a highly metastatic, highly MSH-responsive mouse melanoma cell subline (B16-F10C23) were determined using a radiolabeled, biologically active preparation of the superpotent alpha-MSH analogue, [Nle4,D-Phe7]-alpha-MSH (125I-NDP-MSH). A single high-affinity class of binding site was detected (Kd for NDP-MSH, 5.6 x 10(-11) M; Kd for alpha-MSH, 2.6 x 10(-9) M as determined by Scatchard analysis and heterologous inhibition assays, respectively). alpha-MSH showed nearly identical concentration-response relationships in the radioreceptor assay (inhibition of 125I-NDP-MSH binding) and a bioassay (stimulation of intracellular cyclic AMP accumulation). Furthermore, the respective potencies of three melanotropins, NDP-MSH, alpha-MSH, and adrenocorticotropic hormone, in binding and biological assays were highly correlated. These results indicate that the 125I-NDP-MSH binding site represents the functional MSH receptor. Tumors were induced by inoculation of C57BL/6 mice with B16-F10C23 cells, and the presence of 125I-NDP-MSH binding sites was determined by in situ radiolabeling of frozen tissue sections followed by autoradiography. Specific MSH binding sites were distributed throughout the tumor tissue, but not in associated fibrovascular elements or in neighboring nonmelanoma tissues. As in cultured B16-F10C23 cells, melanotropins inhibited 125I-NDP-MSH binding to tissue sections in a concentration-dependent manner. These results support the hypothesis that functional MSH receptors are expressed in melanoma in situ, suggesting that the activities of melanoma cells in vivo may be subject to modulation by endogenous melanotropins. The methods described will be applicable for studies of the expression and regulation of MSH receptors in human melanoma and other target tissues.

The Molecular Registry of Pituitary Adenomas (REMAH): A bet of Spanish Endocrinology for the future of individualized medicine and translational research. / El Registro Molecular de Adenomas Hipofisarios (REMAH): una apuesta de futuro de la Endocrinología española por la medicina individualizada y la investigación traslacional.

Pituitary adenomas are uncommon, difficult to diagnose tumors whose heterogeneity and low incidence complicate large-scale studies. The Molecular Registry of Pituitary Adenomas (REMAH) was promoted by the Andalusian Society of Endocrinology and Nutrition (SAEN) in 2008 as a cooperative clinical-basic multicenter strategy aimed at improving diagnosis and treatment of pituitary adenomas by combining clinical, pathological, and molecular information. In 2010, the Spanish Society of Endocrinology and Nutrition (SEEN) extended this project to national level and established 6 nodes with common protocols and methods for sample and clinical data collection, molecular analysis, and data recording in a common registry (www.remahnacional.com). The registry combines clinical data with molecular phenotyping of the resected pituitary adenoma using quantitative real-time PCR of expression of 26 genes: Pituitary hormones (GH-PRL-LH-FSH-PRL-ACTH-CGA), receptors (somatostatin, dopamine, GHRH, GnRH, CRH, arginine-vasopressin, ghrelin), other markers (Ki67, PTTG1), and control genes. Until 2015, molecular information has been collected from 704 adenomas, out of 1179 patients registered. This strategy allows for comparative and relational analysis between the molecular profile of the different types of adenoma and the clinical phenotype of patients, which may provide a better understanding of the condition and potentially help in treatment selection. The REMAH is therefore a unique multicenter, interdisciplinary network founded on a shared database that provides a far-reaching translational approach for management of pituitary adenomas, and paves the way for the conduct of combined clinical-basic innovative studies on large patient samples.

Enhancement of gene delivery by an analogue of alpha-MSH in a receptor-independent fashion.

In order to transfect melanoma specifically by receptor-mediated endocytosis we prepared dioctadecyl aminoglycylspermine (lipospermine)--DNA complexes with [Nle(4),D-Phe(7)]-alpha-MSH(4--10), a pseudo-peptide analogue of alpha-melanocyte stimulating hormone (alpha-MSH) linked to a thiol-reactive phospholipid. With these complexes we obtained an up to 70-fold increase of transfection with B16-F1 melanoma cells. However when B16-G4F, an alpha-MSH receptor negative melanoma cell line was transfected, an up to 700-fold increased transfection efficiency was observed. The peptide hormone analogue was equally efficient when it was only mixed with lipospermine--DNA complexes without covalent coupling. In addition to melanoma cells we also obtained up to 30-fold increased transfection with BN cells (embryonic liver cells). Our data show that an alpha-MSH analogue increased transfection independently of the MSH receptor expression but reaches efficiencies approaching those obtained with peptides derived from viral fusion proteins. The absence of targeting of constructs containing [Nle(4),D-Phe(7)]-alpha-MSH(4-10) can probably be attributed due to the relatively modest number of MSH receptors at the surface of melanoma. We suggest, however, that the peptide hormone analogue used in this study has membrane-active properties and could be of interest as helper agent to enhance non-viral gene delivery presumably by endosomal-destabilizing properties.

Identification of the salmon somatolactin receptor, a new member of the cytokine receptor family.

Somatolactin (SL) is a pituitary hormone of the GH/prolactin (PRL) family that so far has been found only in fish. Compared with GH and PRL, the primary structure of SL is highly conserved among divergent fish species, suggesting it has an important function and a discriminating receptor that constrains structural change. However, SL functions are poorly understood, and receptors for SL have not yet been identified. During cloning of GH receptor cDNA from salmon, we found a variant with relatively high (38-58%) sequence identity to vertebrate GH receptors and low (28-33%) identity to PRL receptors; however, the recombinant protein encoding the extracellular domain showed only weak binding of GH. Ligand binding of the recombinant extracellular domain for this receptor confirmed that the cDNA encoded a specific receptor for SL. The SL receptor (SLR) has common features of a GH receptor including FGEFS motif, six cysteine residues in the extracellular domain, a single transmembrane region, and Box 1 and 2 regions in the intracellular domain. These structural characteristics place the SLR in the cytokine receptor type I homodimeric group, which includes receptors for GH, PRL, erythropoietin, thrombopoietin, granulocyte-colony stimulating factor, and leptin. Transcripts for SLR were found in 11 tissues with highest levels in liver and fat, supporting the notion that a major function of SL is regulation of lipid metabolism. Cloning SLR cDNA opens the way for discovery of new SL functions and target tissues in fish, and perhaps novel members of this receptor family in other vertebrates.

Regulation of the rat proopiomelanocortin gene expression in AtT-20 cells. II: Effects of the pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal polypeptide.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP), members of the glucagon-secretin family, have recently been suggested to be involved in the regulation of corticotropin (ACTH) secretion. In this study, we examined the effects of both peptides on POMC gene expression. Using AtT20PL, a clone of the AtT20 mouse corticotroph tumor cells stably transfected with 0.7 kb of the rat POMC 5' promoter-luciferase fusion gene, the effects of both peptides on the POMC promoter activity were estimated by a luciferase assay. PACAP stimulated POMC 5' promoter activity as well as cAMP generation and ACTH secretion in a dose- and time-dependent manner, with the maximal effect being observed 3 h after the start of incubation. A similar effect was observed with VIP. Although the combined effects of PACAP/CRH or VIP/CRH were greater than that of either hormone alone, no such effect was observed between PACAP and VIP. Furthermore, RT-PCR analysis showed the presence of only the PVR3 receptor subtype in this cell line, which is known to have a similar affinity to PACAP and VIP, indicating that both peptides exert their effects through the same receptor. In contrast to the effect of CRH, which was completely abolished by a protein kinase A inhibitor H89, the effects of PACAP/VIP on POMC expression persisted during H89 treatment, suggesting the involvement of alternative intracellular signaling pathway(s) distinct from the protein kinase A system. Our results suggest that PACAP and VIP have positive effects on POMC gene expression and that multiple signaling pathways are involved in the transcriptional event.

Pituitary adenylate cyclase-activating polypeptide receptors of types I and II and glucagon-like peptide-I receptors are expressed in the rat medullary carcinoma of the thyroid cell line 6/23.

The expression of the messenger RNAs coding for glucagon-like peptide-I (GLP-I) receptor, VIP receptor, and pituitary adenylate cyclase-activating polypeptide (PACAP) receptor as well as the expression of the receptor proteins were demonstrated in the rat medullary carcinoma of thyroid cell line 6/23 by the following experiments: 1) RNA extraction, reverse transcriptase, and polymerase chain reaction with specific primers; 2) binding of the radiolabeled ligands [125I]GLP-I-(7-36)-NH2, [125I]PACAP-(1-27), and [125I]VIP and inhibition by, respectively, unlabeled GLP-I-(7-36)-NH2, PACAP-(1-27), and VIP; and 3) study of adenylate cyclase activation by the peptides and selective inhibition of the VIP/PACAP response by the antagonist [D-Phe2]VIP. Besides the highly selective GLP-I receptor, PACAP receptors of types I and II were present on the cell line and coupled to adenylate cyclase. PACAP stimulated the adenylate cyclase through type I and II receptors, whereas VIP interacted with type II receptors only. Messenger RNA analysis indicated that at least three splice variants of the PACAP type I receptor may be expressed in 6/23 cells.

Pituitary adenylate cyclase-activating polypeptide potentiation of Ca2+ entry via protein kinase C and A pathways in melanotrophs of the pituitary pars intermedia of rats.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to stimulate melanotroph secretion, and PACAP-like immunoreactivity and expression of PACAP type I receptor messenger RNA have been identified in the pituitary pars intermedia (PI). The present study showed that PACAP messenger RNA is also expressed in the PI. To examine the mechanism of PACAP action in the PI, cytosolic Ca2+ concentrations ([Ca2+]i) and ionic currents were measured in acutely dissociated rat melanotrophs. In about 40% of the melanotrophs studied, PACAP induced an increase in [Ca2+]i, which was suppressed by extracellular Ca2+ removal; extracellular Na+ replacement; the blocker of L-type Ca2+ channels, nicardipine; or the secreto-inhibitory neurotransmitter, dopamine. The PACAP-induced [Ca2+]i increase was mimicked by activators of protein kinase A (PKA) and protein kinase C (PKC), Sp-diastereomer of cAMP and 1-oleoyl-2-acetyl-sn-glycerol, and was reduced by inhibitors of PKA and PKC, Rp-diastereomer of cAMP and staurosporine. Patch-clamp analysis revealed that PACAP caused inward currents with a reversal potential of -0.8 mV and facilitated voltage-dependent Ba2+ currents. It further revealed that PACAP-induced inward currents were mimicked by 1-oleoyl-2-acetyl-sn-glycerol and inhibited by staurosporine, and that Sp-diastereomer of cAMP facilitated Ba2+ currents. These results suggest that PACAP potentiates Ca2+ entry mechanisms of rat melanotrophs by activation of nonselective cation channels via PKC and facilitation of voltage-dependent Ca2+ channels via PKA.

Receptors for pituitary adenylate cyclase-activating peptide in human liver.

The presence as well as the pharmacological, molecular, and functional properties of pituitary adenylate cyclase-activating peptide (PACAP) receptors have been analyzed in human liver membranes compared in parallel with vasoactive intestinal peptide (VIP) receptors. [125I]PACAP-27 bound to two classes of receptors with high [dissociation constant (Kd) = 0.47 nmol/L] and low (Kd = 8.0 nmol/L) affinities that represented about 34% and 66% of total binding sites, respectively. The pharmacological profile of [125I]VIP and [125I]PACAP-27 binding to membranes supported the coexistence with VIP receptors of specific receptors for PACAP with a mol wt equal to 67.7K. When [125I]PACAP-27 was used, the order of potency of various related peptides for competition of tracer binding was PACAP-27 greater than PACAP-38 greater than VIP. Both PACAP-27 and VIP stimulated adenylate cyclase activity with similar efficacy, although PACAP-27 showed a potency (half-maximal efficient concentration or EC50 = 0.5 nmol/L) greater than that of VIP (EC50 = 4.1 nmol/L). When the two peptides were present simultaneously in the incubation medium, no additive effect on the stimulation of adenylate cyclase activity was observed, which suggests a unique receptor coupled to this enzyme.

alpha-Melanocyte-stimulating hormone induces cell death in mast cells: involvement of NF-kappaB.

Mast cells play a major role in the initiation of inflammation and allergic reactions. As cell numbers are tightly controlled by the interplay of factors affecting cell proliferation, development, and death the regulation of mast cell number may be important. Melanocyte-stimulating hormone inhibits most forms of inflammation by an unknown mechanism. In the present study, we have found that the alpha-melanocyte-stimulating hormone (alpha-MSH) inhibited endotoxin-mediated nuclear transcription factor kappaB (NF-kappaB) activation in different cells correlated with the expression of alpha-MSH receptors. We have also found for the first time that it induces cell death alone or in endotoxin-stimulated mast cells. alpha-MSH-mediated apoptosis was not observed in NF-kappaB overexpressed cells. The inhibitory effect of alpha-MSH was mediated through generation of cAMP, as inhibitors of adenylate cyclase and of protein kinase A reversed its inhibitory effect. Overall, our results suggest that NF-kappaB is the key molecule involved in alpha-MSH-mediated cell death and this may help to regulate mast cell-mediated inflammation.

Expression of VIP and/or PACAP receptor mRNA in peptide synthesizing cells within the suprachiasmatic nucleus of the rat and in its efferent target sites.

The suprachiasmatic nucleus (SCN) contains the predominant circadian pacemaker in mammals. Considerable evidence indicates that VPAC(2) and PAC(1), receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP), play critical roles in maintaining and entraining circadian rhythms. Retinal projections to the rat SCN contain PACAP and terminate mostly in the ventral SCN, the site of VIP neurons. The incidence of VPAC(2) and PAC(1) mRNAs within distinct neuronal populations of the rat SCN has been determined using double-label in situ hybridization. VPAC(2) mRNA was detected in almost all arginine-vasopressin (AVP) neurons of the dorsomedial SCN and in 41% of the VIP neurons; somatostatin (SST) neurons, predominantly in dorsomedial and intermediate regions, showed a decreased incidence (23%). PAC(1) mRNA was present in nearly half of the VIP and SST neurons (45% and 40%, respectively) and in one-third of the AVP neurons (32%). Cells expressing VPAC(2) mRNA also were detected in diencephalic areas that receive VIP-immunoreactive SCN efferents, such as the peri-suprachiasmatic region, lateral subparaventricular zone, parvocellular hypothalamic paraventricular subdivisions, dorsomedial hypothalamic nucleus, and anterior thalamic paraventricular and paratenial nuclei. The extensive distribution of PAC(1) mRNA within the SCN suggests that actions of PACAP are not restricted to the predominantly retinorecipient region. The presence of VPAC(2) mRNA in nearly half the VIP neurons, in almost all the AVP neurons, and at sites receiving VIP-immunoreactive SCN efferents suggests that the SCN VIP neurons are coupled and/or autoregulated and also influence the AVP-containing dorsomedial SCN and distal sites via VPAC(2).

Beta-endorphin and corticotropin immunoreactivity and specific binding in the neuromuscular system of obese-diabetic mice.

Immunoreactivity for two derivatives of pro-opiomelanocortin, beta-endorphin and alpha-melanocortin (or corticotropin), was demonstrated, using a conventional immunoperoxidase method, in some of the intramuscular nerves in muscle sections from obese diabetic (ob/ob) mice and homozygous lean (+/+) mice. The endplate regions were visualized in the sections by staining for acetylcholinesterase reaction product. The proportion of muscle endplates with beta-endorphin-immunoreactive motor nerves was approximately 2.5-fold higher in soleus and extensor digitorum longus muscles and approximately 1.5-fold higher in the diaphragm of the obese (ob/ob) mice compared to the normal lean mice. The proportion of muscle endplates with alpha-melanotropin-immunoreactive motor nerves was between 30 and 53% lower, depending on the muscle type, in the ob/ob mice compared to the lean mice. The muscles of ob/ob and lean mice were investigated for the presence of specific binding sites for [125I]beta-endorphin and for [125I]corticotropin, using autoradiography. Some muscle fibres in soleus, extensor digitorum longus and diaphragm in both the ob/ob and the lean mice exhibited specific binding sites for the radioactive ligands. The binding sites were distributed over the entire surface in these muscle fibres. In the ob/ob mice the number of muscle fibres with specific [125I]beta-endorphin binding sites was six-fold higher in soleus and approximately 10-fold higher in extensor digitorum longus and diaphragm, than in the corresponding muscles of the lean mice. In contrast, the number of muscle fibres with specific [125I]corticotropin binding sites was similar in obese (ob/ob) and lean mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological, molecular and functional characterization of vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide receptors in the rat pineal gland.

Melatonin secretion from the mammalian pineal gland is strongly stimulated by noradrenaline and also by vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Three types of receptors for VIP and PACAP have been characterized so far: VIP1/PACAP receptors and VIP2/PACAP receptors, which possess similar high affinities for VIP and PACAP, and PACAP1 receptors which exhibit a 100-1000-fold higher affinity for PACAP. The aim of the present study was to characterize the receptor subtype(s) mediating the stimulatory effects of VIP and PACAP on melatonin synthesis in the rat pineal gland. Autoradiographic studies showed that PACAP and VIP were equally potent in displacing binding of radioiodinated PACAP27 from pineal sections. Amplification of pineal complementary DNAs by polymerase chain reaction using specific primers for the different receptor subtypes revealed that all three receptor messenger RNAs are expressed and that VIP1/PACAP receptor messenger RNA was predominant over VIP2/PACAP receptor messenger RNA. In vitro, VIP and PACAP stimulated melatonin synthesis with similar high potency and the effect of the two peptides were not additive. The selective VIP1/PACAP receptor agonists [R16]chicken secretin (1-25) and [K15, R16, L27]VIP(1-7)/growth hormone releasing factor(8-27) were significantly more potent than the selective VIP2/PACAP receptor agonist RO 25-1553 in stimulating melatonin secretion. The stimulatory effects of VIP and PACAP were similarly inhibited by the VIP1/PACAP antagonist [acetyl-His1, D-Phe2, K15, R16, L27]VIP(3-7)/growth hormone releasing factor(8-27). These data strongly suggest that VIP and PACAP exert a stimulatory effect on melatonin synthesis mainly through activation of a pineal VIP1/PACAP receptor subtype.

Adaptation of aequorin functional assay to high throughput screening.

AequoScreen, a cellular aequorin-based functional assay, has been optimized for luminescent high-throughput screening (HTS) of G protein-coupled receptor (GPCRs). AequoScreen is a homogeneous assay in which the cells are loaded with the apoaequorin cofactor coelenterazine, diluted in assay buffer, and injected into plates containing the samples to be tested. A flash of light is emitted following the calcium increase resulting from the activation of the GPCR by the sample. Here we have validated a new plate reader, the Hamamatsu Photonics FDSS6000, for HTS in 96- and 384-well plates with CHO-K1 cells stably coexpressing mitochondrial apoaequorin and different GPCRs (AequoScreen cell lines). The acquisition time, plate type, and cell number per well have been optimized to obtain concentration-response curves with 4000 cells/well in 384-well plates and a high signal:background ratio. The FDSS6000 and AequoScreen cell lines allow reading of twenty 96- or 384-well plates in 1 h with Z' values of 0.71 and 0.78, respectively. These results bring new insights to functional assays, and therefore reinforce the interest in aequorin-based assays in a HTS environment.

Immunological localisation of melanocortin 1 receptor on the cell surface of WM266-4 human melanoma cells.

The localisation of melanocortin 1 receptor (MC1R) in WM266-4 human melanoma cells was investigated by applying an antipeptide antiserum specific for the cloned human MC1R (MSH receptor). In enzyme-linked immunosorbent assay (ELISA), the immunoreactivity was detected in the membrane fraction of WM266-4 cells. The ELISA reactivity could be inhibited by an antiserum pre-absorbed with its specific synthetic peptide. In immunocytochemistry, the specific immunoreactivity was demonstrated on the surface of the cells by using either biotin-avidin immunoalkaline phosphatase- or TRITC-staining method. These results indicate that the MC1R is prominently present on the plasma membrane of WM266-4 human melanoma cells.

PACAP(6-38) inhibits the growth of prostate cancer cells.

The effects of pituitary adenylyl cyclase activating polypeptide (PACAP) analogs on prostate cancer cell lines was investigated. 125I-PACAP-27 bound with high affinity to PC-3 cells (Kd = 10 nM) to a single class of sites (Bmax = 30000/cell). By RT-PCR, a major 305 bp band was observed using cDNA derived from PC-3, LNCaP or DU-145 cells. Specific 125I-PACAP binding was inhibited with high affinity by PACAP-27, PACAP-38 and PACAP(6-38) (IC50 values of 15, 10 and 300 nM, respectively) but not by PACAP(28-38). PACAP elevated cAMP and the increase caused by PACAP-27 was reversed by PACAP(6-38). PACAP transiently increased c-fos gene expression and the increase in c-fos mRNA was reversed by PACAP(6-38). PACAP-27 stimulated colony formation in PC-3 cells, whereas PACAP(6-38) reduced colony number and size. In nude mice bearing PC-3 xenografts, PACAP(6-38) significantly slowed tumor growth. These data suggest that biologically active type 1 PACAP receptors are present on human prostate cancer cells and that prostate cancer cell growth is inhibited by PACAP(6-38).

Ontogeny of ACTH(1-24) receptors in rat adrenal glands during the perinatal period.

Binding of ACTH to receptors was studied on crude adrenal membranes from fetal and newborn rats. 125I-Labelled ACTH(1-24) was used as the radioligand, the steroidogenic potency of which was 100-fold lower than that of unlabelled ACTH(1-24). Binding was specific, rapidly equilibrated and temperature dependent. Scatchard analysis of the binding data revealed a single class of binding sites with a dissociation constant of about 100 nmol/l at all stages of development studied. The concentration of ACTH receptors expressed per mg membrane proteins decreased in fetuses between days 17 and 21 of gestation and remained stable in newborn rats from weeks 1 to 4. The number of ACTH receptors expressed per adrenal increased regularly in fetal and newborn rats. The perinatal evolution of these concentrations of ACTH receptors is related to the increase in the size of the adrenals and the changes in cytoplasmic structures of the adrenocortical cells. When the number of ACTH-binding sites was expressed per microgram DNA, maximum values occurred in fetuses on day 19 of gestation, and minimum values in newborn rats, 1 week after birth. There was an excellent correlation between the plasma levels of immunoreactive ACTH and corticosterone and the number of ACTH receptors per microgram DNA during the perinatal period. Other results suggest that ACTH is able to up-regulate the number of its own receptors.

Presynaptic localization of the PACAP-typeI-receptor in hippocampal and cerebellar mossy fibres.

The distribution of PACAP-typeI-receptor (PACAP-I-R) mRNA and protein was studied in mouse using probes and a newly developed antiserum recognizing all known splice variants. RNase protection assays revealed highest expression levels of PACAP-I-R mRNA in brain, in particular the hypothalamus and hippocampus. At the cellular level, in situ hybridization analysis demonstrated widespread distribution of PACAP-I-R mRNA in neurons throughout the brain, while glial cells did not express the gene. Highest expression levels of PACAP-I-R mRNA were observed in three regions: the limbic system, the hypothalamus, and the brainstem. In accordance with data obtained from in situ hybridization analysis, immunohistochemistry showed widespread distribution of PACAP-I-R like immunoreactivity in the neuropil. Rather strong immunoreactivity was found in cerebellar and hippocampal mossy fibres where double immunolabelling revealed the presynaptic localization of the receptor protein. At the ultrastructural level, PACAP-I-R like immunoreactivity was observed around synaptic vesicles and close to the presynaptic grid in hippocampal mossy fibre terminals. This finding is in contradiction to the described postsynaptic localization of the PACAP-I-R in dendritic processes of hippocampal granule cells in rat. Due to their presynaptic induction, mossy fibre LTPs are distinctly different from LTPs in all other hippocampal regions. Therefore, the presynaptic localization of the PACAP-I-R in mossy fibre terminals may implicate this gene in influencing the synaptic strength of the mossy fibre pathway and hence memory consolidation.

Delayed up-regulation of Zac1 and PACAP type I receptor after transient focal cerebral ischemia in mice.

The Zac1 gene encodes a zinc finger protein that regulates both apoptosis and cell cycle arrest in vitro. Furthermore, Zac1 protein seems to trans-activate the gene encoding the type I receptor for pituitary adenylate cyclase activating polypeptide (PACAP). Northern blot analysis revealed high levels of Zac1 mRNA in the rodent brain. In the present study, we demonstrate by in situ hybridization histochemistry a progressive increase in Zac1 transcripts in the mouse brain from day 1 to day 3 following transient focal cerebral ischemia. Moreover, we observed an up-regulation of PACAP type I receptor mRNA expression showing a similar temporospatial distribution. Late induction of cell death promoting Zac1 in the post-ischemic brain may be attributed to delayed or secondary cell death. Co-induction of the type I receptor for neurotrophic PACAP however, points to a role in restorative processes.

A critical view of the methods for characterization of the VIP/PACAP receptor subclasses.

The binding properties of the three cloned VIP/PACAP receptors and their coupling to G proteins and effectors can be studied in cells expressing each recombinant protein. The data obtained in these models must be critically evaluated: the expression of a high receptor density may reveal irrelevant receptors states and coupling to non-cognate G protein, and entail a marked amplification of the response as well as distortions in the selectivity profile of full and partial agonists. These models are, however, of great interest in the design of selective agonists and antagonists for each receptor subtype. The availability of selective ligands will facilitate the identification of the receptor subtype responsible for PACAP and VIP actions in cells and tissues.